Cytotoxic T cells swarm by homotypic chemokine signalling
- EMBL Australia, Single Molecule Science node, University of New South Wales, Australia
- School of Medical Sciences, Faculty of Medicine, University of New South Wales, Australia
- Sydney Medical School, The University of Sydney, Australia
- The Walter and Eliza Hall Institute of Medical Research, Australia
- Department of Medical Biology, University of Melbourne, Australia
- Children's Cancer Research Unit, The Children's Hospital at Westmead, Australia
- The Kirby Institute for Infection and Immunity in Society, UNSW, Australia
- Neuroscience Research Australia (NeuRA), Australia
- Department of Statistics and Actuarial Science, University of Waterloo, Canada
- Discipline of Child and Adolescent Health, University of Sydney, Australia
- Biofab3D, St. Vincent’s Hospital, Australia
- Institute for Innovation in Mental and Physical Health and Clinical Translation (iMPACT), School of Medicine, Deakin University, Australia
- Advanced Biomaterials Lab, Research School of Engineering, ANU, Australia
- School of Computer Science, Westmead Initiative, and Charles Perkins Centre, University of Sydney, Australia
Figures
Figure 1 with 1 supplement
CTLs swarm as they attack tumour masses.
(A) Schematic of ex vivo tumouroid model: a high-density mass of tumour cells and collagen (tumouroid, magenta) is surrounded by a 3D collagen matrix containing dispersed CTLs (green) in a closed …
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Figure 1—source data 1
Source data file for Figure 1.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
In silico model of CTL recruitment towards sites of tumour engagement.
(A) Schematic of bead tumouroid model: a high-density mass of collagen and beads blue, either uncoated or coated with cognate H-2Kb/SIINFEKL is surrounded by a 3D collagen matrix containing …
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Figure 1—figure supplement 1—source data 1
Source data file for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig1-figsupp1-data1-v3.xlsx
Figure 2 with 1 supplement
CTLs migrate rapidly and directionally towards sites of cognate target engagement due to a diffusing homotypic signal.
(A) Schematic of assay enabling 3D tracking of CTL movements relative to a tumouroid exposing a straight interface (scale bar: 50 µm). (B) Average speed and forward migration index (FMI) of CTLs …
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Figure 2—source data 1
Source data file for Figure 2.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
CTLs transmigrate towards cognate supernatants.
(A) Schematic illustration of the transmigration assay. CTLs were co-incubated with SIINFEKL-pulsed EL4 tumour cells or unpulsed non-cognate EL4 cells for 3 hr. The supernatant was filtered and used …
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Figure 2—figure supplement 1—source data 1
Source data file for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig2-figsupp1-data1-v3.xlsx
Figure 3 with 1 supplement
Engagement of cognate tumouroid gives rise to a diffusing wave of chemoattraction.
(A) Confocal images showing the distribution of CTLs (green) in relation to a SIINFEKL-pulsed EL4 cognate tumouroid at 0 and 2 hr. Pseudocolour: distance to nearest cell in µm. (B) Cumulative …
Figure 3—figure supplement 1
Long-range attraction assay in a non-cognate setting.
(A) Left: Schematic illustration showing CTL movements up to 1.6 mm away from the tumouroids comprising EL4 cells. Right: Representative confocal imaging showing the distribution of CTLs in relation …
Figure 4 with 4 supplements
CCL3 and CCL4 secretion drives T cell swarming.
(A) Average speed (Left) and FMI (Right) of OT1 CTLs adjacent to non-cognate EL4 or cognate SIINFEKL-pulsed EL4 tumouroids with pre-embedded tumour-reactive CTLs not treated with pertussis toxin …
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Figure 4—source data 1
Source data file for Figure 4.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
CCL3 and CCL4 are upregulated and secreted by activated CTLs and mediate homotypic recruitment.
(A, B) Quantification of chemokine mRNA levels by qRT-PCR in OT1 (A) or gBT1 (B) CTLs exposed to H-2Kb/SIINFEKL cognate beads, or cognate SIINFEKL- or SSIEFARL-pulsed EL4 respectively, or …
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Figure 4—figure supplement 1—source data 1
Source data file for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig4-figsupp1-data1-v3.xlsx
Figure 4—figure supplement 2
CTLs engaging cognate targets generate long-range chemokine gradients.
(A) Schematic illustration of experiment with cytometric bead array (CBA) beads embedded in collagen matrix adjacent to a cognate SIINFEKL-pulsed EL4 tumouroid co-embedded with CTLs. The CBA beads …
Figure 4—figure supplement 3
CCL3/CCL4-secreting tumour cells induce swarming and tumour infiltration.
(A) Flow cytometry analysis of EL4 tumour cells that constitutively secrete CCL3-mScarletI and CCL4-miRFP670. (B) Quantification of absolute CCL3 (right) and CCL4 (left) concentration detected in …
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Figure 4—figure supplement 3—source data 1
Source data file for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig4-figsupp3-data1-v3.xlsx
Figure 4—figure supplement 4
Leukocyte tumour infiltration in vivo.
(A) Rag-/- mice were engrafted subcutaneously with EL4 or CCL3/4-secreting EL4 tumour cells in contralateral flanks. On day 7 post-engraftment, 5 × 106 OT1GFP CTLs were transferred intravenously. …
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Figure 4—figure supplement 4—source data 1
Source data file for Figure 4—figure supplement 4.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig4-figsupp4-data1-v3.xlsx
Figure 5 with 3 supplements
T cell swarming is CCR5-dependent.
(A) FMI of polyclonal WT or Ccr5-/- T cells adjacent to non-cognate EL4 or cognate SIINFEKL-pulsed EL4 tumouroids pre-embedded with tumour-reactive CTLs. Box-whiskers indicate medians and the …
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Figure 5—source data 1
Source data file for Figure 5.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Chemokine receptor CCR5 mediates homotypic recruitment of CTLs.
(A) qRT-PCR analysis of chemokine receptor expression in OT1 CTLs. Data points from four independent experiments; red bars indicate mean. Data is normalised to universal mouse cDNA (positive …
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Figure 5—figure supplement 1—source data 1
Source data file for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig5-figsupp1-data1-v3.xlsx
Figure 5—figure supplement 2
In vivo tumour homing and rejection.
(A) WT and Ccr5-/- OT1 CTLs were adoptively transferred into Rag-/- mice engrafted with control and CCL3-mScarletI/CCL4-miRFP670 secreting non-cognate tumours in contralateral flanks. Tumours were …
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Figure 5—figure supplement 2—source data 1
Source data file for Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig5-figsupp2-data1-v3.xlsx
Figure 5—figure supplement 3
Intravital imaging following local intratumoural injection of CCL3.
(A) Schematic representation of the intravital imaging set-up allowing for intratumoural delivery of CCL3-containing hydrogel into the centre of a field of view with a side of ~3 mm recording CTL …
Figure 6 with 1 supplement
Homotypic recruitment of human polyclonal and CAR T cells.
(A) Transmigration of human T cells towards supernatant from T cells conjugated with IgG-coated beads (control beads), recombinant human CCL3 and CCL4, or supernatant from T cells conjugated with …
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Figure 6—source data 1
Source data file for Figure 6.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Chemokine expression in activated human polyclonal T cells, and CAR T cell sorting and cytotoxicity.
(A) qRT-PCR analysis of chemokine expression in human T cells exposed to activating (αCD3/CD28-coated) beads relative to control (IgG-coated) beads. (B) Primary human T cells were transduced with …
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Figure 6—figure supplement 1—source data 1
Source data file for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/56554/elife-56554-fig6-figsupp1-data1-v3.xlsx
Figure 7
Model of multistate mass recruitment of CTLs to a target site.
‘Searcher’ CTLs migrate randomly in search of target cells. When they recognise targets, they become ‘engager/recruiter’ CTLs that secrete CCL3 and CCL4. CCR5-expressing CTLs within range migrate …
Author response image 1
mRNA expression levels of various chemokines in B16 tumour cells relative to EL4 tumour cells as measured by qPCR.
Videos
Video 1
CTLs swarm around a cognate tumouroid.
(Total view) Long-term time-lapse imaging (40 hr) of OT1GFP CTLs (dark) embedded in 3D collagen matrix around cognate EL4 tumouroid (light grey disc). (Inset view) Brightfield (BF) and fluorescence …
Video 2
CTLs infiltrate deep into cognate tumour masses embedded with tumour-reactive CTLs.
Long-term (16 hr) time-lapse imaging of OT1 CTLs (green) embedded in collagen matrix around EL4 tumouroid (magenta). (Left) Non-cognate tumouroid; no significant accumulation of CTLs at tumouroid. …
Video 3
Tumour-reactive CTLs actively eliminate cognate tumour cells within the tumouroid.
3D time-lapse (3 hr) imaging of OT1 CTLs (green) embedded in a tumouroid composed of cognate tumour cells (purple) and dense collagen matrix. Propidium iodide was added to the medium to detect lysed …
Video 4
In silico simulations of CTL movements around cognate tumouroid as agent-based processes.
Motility models bootstrapped from experimental data. Gray: tumouroid; green: agents migrating without influence of chemoattractive signal; blue: agents encountering chemoattractant concentrations …
Video 5
Tracking individual CTL movements in 3D at high temporal resolution.
3D timelapse (1 hr) imaging of OT1 CTLs (green) embedded in collagen matrix near cognate tumouroid exposing straight interface (out of view to the left) with embedded tumour-reactive CTLs. Track …
Video 6
Tracking individual CTL movements up to 2 mm from tumouroid.
3D time-lapse (2 hr) imaging of OT1 CTLs (green) embedded in collagen matrix adjacent to a tumouroid (magenta). (Top) Non-cognate tumouroid. (Bottom) Cognate tumouroid with embedded tumour-reactive …
Video 7
Intravital imaging reveals CTL recruitment to site of intratumoural CCL3 injection.
(Injection Process) 2D time-lapse imaging of intratumoural injection of shear-thinning hydrogel containing recombinant CCL3 and Dextran-AF647 (white). (Injection Site) 2D fluorescence confocal image …
Video 8
Activated primary human T cells engage in homotypic recruitment.
Long-term (14 hr) time-lapse imaging of primary human T cells (green) embedded in collagen around bead-stimulated human T cells (magenta). (Left) Control (IgG-coated) beads. (Right) Activating …
Video 9
CAR T cells engaging antigen-expressing glioblastoma tumouroids induce swarming.
Long-term (14 hr) time-lapse imaging of human CAR T cells (green) embedded in collagen matrix around tumouroids (magenta). (Left) Tumouroid contains EphA2-specific CAR T cells and EPhA2-negative …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | Rag-deficient B6.129S7-Rag < tm1mom > JAusb (RAG1) | Australian BioResources | Used for in vivo experiments | |
| Strain, strain background (M. musculus) | B6.SJL-PtprcaPepcb/BoyJAusb (PTPRCA) | Australian BioResources | Used for in vivo experiments | |
| Strain, strain background (M. musculus) | B6.129p2-ccr5 tm1Kuz/J (CCR5-/-) | Australian BioResources | 7–12 week mice used to isolate CCR5KO CTLs | |
| Strain, strain background (M. musculus) | B6-Tg-OTI (OT1) | Australian BioResources | 7–12 week mice used to isolate OT1 CTLs | |
| Strain, strain background (M. musculus) | B6-Tg(CAG-Lifeact/EGFP)-OTI (OT1GFP) | Galeano Niño et al., 2020 | 7–12 week mice used to isolate LifeactGFP OT1 CTLs | |
| Strain, strain background (M. musculus) | B6.gBT-I/uGFP (gBT1-GFP) | Gift from W. R. Heath | 7–12 week mice used to isolate GFP gBT1 CTLs | |
| Strain, strain background (M. musculus) | B6.gBT-I/dsRED (gBT1-dsRED) | Gift from W. R. Heath | 7–12 week mice used to isolate dsRED gBT1 CTLs | |
| Cell line (M. musculus) | EL-4 lymphoma | ATCC | Cat. #: TIB-39 | |
| Cell line (M. musculus) | NIH/3T3 fibroblast | ATCC | Cat. #: CRL-1658 | |
| Cell line (Homo-sapiens) | WK1 glioblastoma cells | Day et al., 2013 | Cultured as described in Stringer et al., 2019 | |
| Cell line (Homo-sapiens) | GP2-293 embryonic kidney cells | Clonetech | Cat. #: 631458 | |
| Cell line (M. musculus) | mCCL3-mScarletI secreting EL4 cells | This paper | EL4 cells transduced with CCL3-mScarletI construct. Sequence available in Materials and methods. | |
| Cell line (M. musculus) | mCCL4-miRFP670 secreting EL4 cells | This paper | EL4 cells transduced with CCL4-miRFP670 construct. Sequence available in Materials and methods. | |
| Cell line (M. musculus) | ‘CCL3/4-secreting EL4’ i.e. mCCL3-mScarletI and mCCL4-miRFP670 secreting EL4 cells | This paper | EL4 cells transduced with CCL3-mScarletI and CCL4-miRFP670 construct. Sequences available in Materials and methods. | |
| Cell line (M. musculus) | EL4.HSV | This paper | EL4 cells transduced with bicistronic SSIEFARL epitope and mTagBFP2. Sequence available in Materials and methods. | |
| Cell line (M. musculus) | EL4.OVA | This paper | EL4 cells transduced with bicistronic SIINFEKL epitope and mTagBFP2. Sequence available in Materials and methods. | |
| Cell line (Homo-sapiens) | CAR T cells | This paper | Primary human T cells transduced with EphA2-specific CAR construct. Produced by Kramer lab at Children's Cancer Research Unit, The Children's Hospital at Westmead. Information on CAR constructs available in Materials and methods. | |
| Transfected construct (M. musculus) | MSCV-based retrovial expression vector (LENC) | Fellmann et al., 2013 Gift from J. Zuber | http://doi.org/10.1016/j.celrep.2013.11.020 | Retrovirus construct to express cDNA |
| Transfected construct (M. musculus) | pMD2.G | Gift from Didier Trono | Addgene plasmid # 12259; RRID:Addgene_12259 | Construct for VSVG pseudotyping retrovirus |
| Transfected construct (M. musculus) | mCCL3-mScarletI gBlocks | Integrated DNA Technologies | gBlocks cloned into LENC | |
| Transfected construct (M. musculus) | mCCL4-miRFP670 gBlocks | Integrated DNA Technologies | gBlocks cloned into LENC | |
| Transfected construct (M. musculus) | SSIEFARL-mTagBFP2 gBlocks | Integrated DNA Technologies | gBlocks cloned into LENC | |
| Transfected construct (M. musculus) | SIINFEKL-mTagBFP2 gBlocks | Integrated DNA Technologies | gBlocks cloned into LENC | |
| Transfected construct (Homo- sapiens) | EphA2-specific CAR construct | Chow et al., 2013 and Yi et al., 2018 | Construct for lentiviral transduction of human T cells | |
| Biological sample (Homo-sapiens) | Peripheral blood mononuclear cells (PBMCs) | Sydney Children’s Hospitals Network | Used to isolate human T cells | |
| Antibody | Anti-XCL1 (polyclonal goat IgG) | R & D Systems | Cat. #: AF486 RRID:AB_2216915 | (5 µg/ml) |
| Antibody | Anti-CCL1 (monoclonal rat IgG2a) | R & D Systems | Cat. #: MAB845 RRID:AB_2070618 | (5 µg/ml) |
| Antibody | Anti-CXCR2 (monoclonal rat IgG2a) | R & D Systems | Cat. #: MAB2164-100 RRID:AB_358062 | (10 µg/ml) |
| Antibody | Anti-CCL3 (polyclonal goat IgG) | R & D Systems | Cat. #: AF-450-NA RRID:AB_354492 | (5 µg/ml) |
| Antibody | Anti-CCL4 (monoclonal rat IgG2a) | R & D Systems | Cat. #: MAB451-100 RRID:AB_2259676 | (5 µg/ml) |
| Antibody | Anti-CXCR4 (monoclonal rat IgG2b) | R & D Systems | Cat. #: MAB21651-100 RRID:AB_2801441 | (10 µg/ml) |
| Antibody | Anti-CCL5 (polyclonal goat IgG) | R & D Systems | Cat. #: AF478 RRID:AB_10080077 | (1 µg/ml) |
| Antibody | Anti-CCL9/10 (monoclonal rat IgG1) | R & D Systems | Cat. #: MAB463 RRID:AB_2259783 | (5 µg/ml) |
| Antibody | Anti-CXCL10 (monoclonal rat IgG2a) | R & D Systems | Cat. #: MAB466 RRID:AB_2292486 | (1 µg/ml) |
| Antibody | Anti-CXC12 (monoclonal mouse IgG1) | R & D Systems | Cat. #: MAB310 RRID:AB_2276927 | (1 µg/ml) |
| Antibody | Anti-CD3ε (monoclonal Armenian Hamster) | BioLegend | Cat. #: 100302 RRID:AB_312667 | (1 µg/ml) |
| Antibody | Anti-CD28 (monoclonal Syrian Hamster) | BioLegend | Cat. #: 102101 RRID:AB_312866 | (1 µg/ml) |
| Antibody | Rat IgG1, κ Isotype control (Rat monoclonal) | BioLegend | Cat. #: 400402 RRID:AB_326508 | (1 µg/ml) |
| Antibody | Mouse IgG1, κ Isotype control (Mouse monoclonal) | BioLegend | Cat. #: 401401 RRID:AB_2801452 | (1 µg/ml) |
| Antibody | APC anti-CD44 (monoclonal rat IgG2b) | BD Biosciences | Cat. #: 553991 RRID:AB_10050405 | (1 µg/ml) |
| Antibody | Pacific Blue anti-CD8a (monoclonal rat IgG2a) | BD Biosciences | Cat. #: 558106 RRID:AB_397029 | (1 µg/ml) |
| Antibody | Biotin anti-CD3ε (monoclonal Armenian Hamster) | Thermo Fisher Scientific | Cat. #: 13-0031-82 RRID:AB_466319 | (1 µg/ml) |
| Antibody | Biotin anti-CD28 (monoclonal Syrian Hamster) | Thermo Fisher Scientific | Cat. #: 13-0281-82 RRID:AB_466411 | (1 µg/ml) |
| Antibody | Biotin anti-CD3 (monoclonal mouse IgG2a) | Thermo Fisher Scientific | Cat. #: 13-0037-82 RRID:AB_1234955 | (1 µg/ml) |
| Antibody | Biotin anti-CD28 (monoclonal mouse IgG1) | Thermo Fisher Scientific | Cat. #: 13-0289-82 RRID:AB_466415 | (1 µg/ml) |
| Antibody | Biotin rat IgG2a κ Isotype Control (Rat polyclonal) | eBioscience | Cat. #: 13-4321-82 RRID:AB_470084 | (1 µg/ml) |
| Antibody | Biotin IgG Isotype Control (Armenian hamster) | eBioscience | Cat. #: 14-4888-81 RRID:AB_470128 | (1 µg/ml) |
| Antibody | Brilliant Violet 711 anti-CD11b (monoclonal rat IgG2b) | BioLegend | Cat. #: 101241 RRID:AB_11218791 | (2 µg/ml) |
| Antibody | Alexa Fluor 488 anti-NK1.1 (monoclonal mouse IgG2a) | BioLegend | Cat. #: 108717 RRID:AB_493184 | (2 µg/ml) |
| Antibody | FITC anti-Ly-6C (monoclonal rat IgG2c) | BioLegend | Cat. #: 128005 RRID:AB_1186134 | (2 µg/ml) |
| Antibody | Brilliant Violet 510 anti-CD90.2 (monoclonal rat IgG2b) | BioLegend | Cat. #: 105335 RRID:AB_2566587 | (2 µg/ml) |
| Antibody | Brilliant Violet 421 anti-CD64 (monoclonal mouse IgG1) | BioLegend | Cat. #: 139309 RRID:AB_AB_2562694 | (2 µg/ml) |
| Antibody | APC/Fire 750 anti-CD45 (monoclonal rat IgG2b) | BioLegend | Cat. #: 103153 RRID:AB_2572115 | (2 µg/ml) |
| Antibody | APC/Fire 750 anti-CD45.1 (monoclonal mouse IgG2a) | BioLegend | Cat. #: 110751 RRID:AB_2629805 | (2 µg/ml) |
| Antibody | FITC anti-CD11c (monoclonal Armenian Hamster) | BioLegend | Cat. #: 117305 RRID:AB_313774 | (2 µg/ml) |
| Antibody | Pacific blue anti-MHC class II (monoclonal rat IgG2b) | BioLegend | Cat. #: 107619 RRID:AB_493528 | (2 µg/ml) |
| Antibody | APC anti-CD8a (monoclonal rat IgG2a) | BioLegend | Cat. #: 100711 RRID:AB_312750 | (2 µg/ml) |
| Antibody | Biotin anti-CCL3 (polyclonal Rabbit sera) | PeproTech | Cat. #: 500-P121BT | (1 µg/ml) |
| Peptide, recombinant protein | OVA257-264 peptide, SIINFEKL | AusPep | Cat. #: 2711 | (1, 0.2, 0.1, 0.02, 0.001 µg/ml) |
| Peptide, recombinant protein | gB498-505 peptide, SSIEFARL | Auspep | Cat. #: gb498-505 | (1 µg/ml) |
| Peptide, recombinant protein | Recombinant mouse IL-2 | BioLegend | Cat. #: 575408 | (100 ng/ml) |
| Peptide, recombinant protein | Recombinant human IL-7 | Miltenyi Biotech Australia | Cat. #: 130-095-362 | (10 ng/ml) |
| Peptide, recombinant protein | Recombinant human IL-15 | Miltenyi Biotech Australia | Cat. #: 130-095-764 | (5 ng/ml) |
| Peptide, recombinant protein | Corning Collagen I Rat Tail Natural | In Vitro Technologies | Cat. #: FAL354236 | (~1.5 mg/ml) |
| Peptide, recombinant protein | Monobiotinylated H-2Kb/SIINFEKL | Tetramer Synthesis Service | John Curtin School of Medical Research | (1 µg/ml) |
| Peptide, recombinant protein | Recombinant human (cross reactive with murine) CCL1 | Peprotech | Cat. #: 300–37 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant Mouse XCL1 | R & D systems | Cat. #: 486-LT-025 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant Murine CCL3 | Peprotech | Cat. #: 250–09 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant Murine CCL4 | Peprotech | Cat. #: 250–32 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant Murine CCL5 | Peprotech | Cat. #: 250–07 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant murine CCL9/10 | Peprotech | Cat. #: 250–12 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant murine CXCL10 | Peprotech | Cat. #: 250–16 | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Recombinant Human CXCL12 | Peprotech | Cat. #: 300-28A | (1, 10 and 100 ng/ml) |
| Peptide, recombinant protein | Lysyl Endopeptidase, Mass Spectrometry Grade (Lys-C) | FUJIFILM Wako Pure Chemical Corp | Cat. #: 121–05063 | (1 µg) |
| Sequence-based reagent | TruSeq Stranded mRNA | Illumina | Cat. #: 20020594 | |
| Chemical compound, drug | CellTracker Orange 5-(and-6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (CMTMR) | Invitrogen | Cat. #: C2927 | (5 µM) |
| Chemical compound, drug | CellTracker Deep Red | Invitrogen | Cat. #: C34565 | (1 µM) |
| Chemical compound, drug | CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) | Invitrogen | Cat. #: C7025 | (5 µM) |
| Chemical compound, drug | Ketamine | Provet Pty Ltd | Cat. #: KETAM I | (100 mg/kg) |
| Chemical compound, drug | Xylazine | Provet Pty Ltd | Cat. #: XYLA Z2 | (15 mg/kg) |
| Chemical compound, drug | Pertussis toxin from Bordetella pertussis (PTX) | Sigma Aldrich | Cat. #: P7208 | (100 ng/ml) |
| Chemical compound, drug | Inactive mutated version of Pertussis toxin from Bordetella pertussis (m-PTX) | Sigma Aldrich | Cat. #: PT-16.0003 | (100 ng/ml) |
| Chemical compound, drug | Maraviroc (CCR5 antagonist) | Sigma Aldrich | Cat. #: PZ0002-5mg | (10 µg/ml) |
| Chemical compound, drug | Cenicriviroc (CCR2 + CCR5 dual inhibitor) | AdooQ Bioscience | Cat. #: A13632-10 (000–22038) | (1 µM) |
| Chemical compound, drug | CCR2 antagonist | Santa Cruz Biotechnology Inc | Cat. #: sc-202525 | (1 µM) |
| Chemical compound, drug | UCB35625 (CCR1 + CCR3 dual inhibitor) | R & D Systems | Cat. #: 2757/1 | (50 µM) |
| Chemical compound, drug | Iodoacetamide (IAM) | Sigma Aldrich | Cat. #: I6125-5G | (1 M) |
| Chemical compound, drug | Dithiothreitol (DTT) | Sigma Aldrich | Cat. #: 43816–10 ML | (50 mM) |
| Commercial assay or kit | EasySep Direct Human T Cell Isolation Kit | Stem Cell Technologies | Cat. #: 19661 | |
| Commercial assay or kit | T Cell TransAct, human | Miltenyi Biotech Australia | Cat. #: 130-111-160 | |
| Commercial assay or kit | EasySep Human CD19 Positive Selection Kit II | Stem Cell Technologies | Cat. #: 17854 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat. #: 74104 | |
| Commercial assay or kit | High-Capacity cDNA Reverse Transcription Kit | ThermoFisher Scientific | Cat. #: 4368814 | |
| Commercial assay or kit | Sandwich ELISA kits (CCL1) | OriGene Technologies | Cat. #: EA100390 | |
| Commercial assay or kit | Sandwich ELISA kits (CCL9) | OriGene Technologies | Cat. #: EA100725 | |
| Commercial assay or kit | Cytometric Bead Array: Multi-Analyte Flow Assay Kit (CCL3, CCL4, CCL5, CXCL10, TNF-α and IFN-γ) | LEGENDplex | Custom mouse panel | |
| Commercial assay or kit | BD Cytometry Beads array Mouse MIP-1b Flex Set (Bead C9) | BD Biosciences | Cat. #: 558343 | |
| Commercial assay or kit | BD Cytometry Beads array Mouse MIP-1a Flex Set (Bead C7) | BD Biosciences | Cat. #: 558449 | |
| Commercial assay or kit | CellTrace CFSE Proliferation Kit | ThermoFisher Scientific | Cat. #: C34554 | |
| Commercial assay or kit | CellTrace Violet Proliferation Kit | ThermoFisher Scientific | Cat. #: C34571 | |
| Commercial assay or kit | Cytofix/Cytoperm Plus Kit (with BD GolgiStop) | BD Bioscience | Cat. #: 554715 | |
| Software, algorithm | FlowJo software version 10.2 | Tree Star Inc | flowjo.com | |
| Software, algorithm | Prism GraphPad Software version 8 | GraphPad | Imaris.oxinst.com | |
| Software, algorithm | Leica Application Suite (LAS) X software version 3.3.0.16794 | Leica Microsystems | Leica-microsystems.com | |
| Software, algorithm | FLUOstaromega software version 5.10 R2 | BMG Labtech GmbH | Bmglabtech.com | |
| Software, algorithm | CFX Manager Software | Bio-Rad | Bio-rad.com | |
| Software, algorithm | BioStation IM software | Nikon | nikon.com | |
| Software, algorithm | ImageJ/FIJI | US National Institutes of Health | Imagej.net | |
| Software, algorithm | Matlab | Mathworks | Mathworks.com | |
| Software, algorithm | R | R Core Team | r-project.org | |
| Software, algorithm | Plot2 for Mac 2.6.1 | Mike Wesemann | apps.micw.org | |
| Software, algorithm | motilisim | Read et al., 2016 | https://github.com/marknormanread/TcellSwarming | |
| Software, algorithm | FastQC (version 0.11.5) | Babraham Institute | bioinformatics.babraham.ac.uk | |
| Software, algorithm | Trimmomatic (version 0.36) | Bolger, A. M., Lohse, M., and Usadel, B. | usadellab.org | |
| Software, algorithm | FeatureCounts (version 1.5.1) | Bioconductor | bioconductor.org | |
| Software, algorithm | STRING DB | ELIXIR | string-db.org | |
| Software, algorithm | MaxQuant (version 1.5.8.3) | Max Plank Insitute of Biochemistry | maxquant.org | |
| Software, algorithm | LFQ Analyst | Monash University | bioinformatics.erc.monash.edu/apps/LFQ-Analyst | |
| Software, algorithm | Limma | Bioconductor | bioconductor.org | |
| Software, algorithm | Motility_analysis package | Python Software Foundation | https://github.com/marknormanread/TcellSwarming | |
| Other | Streptavidin-coated polystyrene particles | Spherotech Inc | Cat. #: SVP-60–5 | 6.0–8.06 µm diameter |
| Other | SPHERO AccuCount blank particles | Spherotech Inc | Cat. #: QACBP-70–10 | |
| Other | Sera-Mag Speed beads | ThermoFisher Scientific | Cat. #: 65152105050250 | |
| Other | Sera-Mag Speed beads | ThermoFisher Scientific | Cat. #: 45152105050250 | |
| Other | Trypsin-gold | Promega | Cat. #: V5280 | |
| Other | Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | Cat. #: D2650-100ml | |
| Other | Collagenase IV | Sigma-Aldrich | Cat. #: C5138-1G | |
| Other | 4’, 6-Diamidino-2-phenylindole (DAPI) | ThermoFisher Scientific | Cat. #: D3571 | (0.5 µg/ml) |
| Other | 10 kDA Dextran labelled with AlexaFluor647 (Dextran-AF647) | ThermoFisher Scientific | Cat. #: D22914 | (10 µg/ml) |
| Other | Fmoc-Asp-Wand resin | GL Biochem | Cat. #: 40501 | |
| Other | 1-hydroxybenzotriazole (HOBt) | GL Biochem | Cat. #: 2592-95-2 | |
| Other | 2-(1H-Benzotriazole-1-yl)−1,1,3,3-Tetramethyluronium hexafluorophosphate (HBTU) | GL Biochem | Cat. #: 94790-37-1 |
Additional files
-
Supplementary file 1
Transcriptomics.
Transcriptomics data of CTLs alone, in indicated conjugations or exposed to indicated supernatants from conjugations with tumour cells. Values are read counts obtained from featureCounts after alignment with TopHat2 to the GRCm38 reference genome.
- https://cdn.elifesciences.org/articles/56554/elife-56554-supp1-v3.xlsx
-
Supplementary file 2
Secretomics.
Tables detailing secreted proteins identified by quantitative mass spectrometry analysis, including the proteins exhibiting significant differences between the cognate versus non-cognate cells and beads.
- https://cdn.elifesciences.org/articles/56554/elife-56554-supp2-v3.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/56554/elife-56554-transrepform-v3.pdf
